1. Field of the Invention
The present invention relates to a process for enzymatic production of peptides. More particularly, the invention relates to a process for producing peptides by using a specific group of enzymes as catalysts.
2. Description of the Prior Art
It is known to carry out syntheses of peptides by more or less sophisticated coupling reactions, both in respect of homogeneous synthesis and of heterogeneous solid phase synthesis. All these chemical methods, however, involve the risk of undesirable secondary reactions and racemizations, and it is therefore necessary to control the chemical reactions carefully to minimize or eliminate these problems. Moreover, the amino acid side chains must often be protected, requiring deblocking as the last chemical step to produce the desired peptides. Depending upon the size of the synthesized peptide, the yields may be low, and the secondary reactions frequently necessitate cumbersome purification procedures to obtain the pure peptide. All these inherent problems of chemical peptide syntheses plus the high price of several of the coupling reagents and the blocking amino acid derivatives mean that even small synthetic peptides, e.g. pentapeptides, are relatively expensive to produce.
Since enzymes are very specific catalysts, and proteolytic enzymes are thus able to hydrolyze peptide bonds in proteins, studies have previously been made of the possibilities of reversing this hydrolysis reaction or, in other words, of utilizing enzymes as catalysts in the synthesis of peptide bonds. Bergmann and Fraenkel-Conrat (ref. 1) and Bergmann and Fruton (ref. 2) made a detailed examination of this and showed in 1937 that papain and chymotrypsin could catalyze the coupling of certain acylamino acids and amino acid anilides. These studies have been continued and intensified on the basis of two fundamentally different approaches, viz. a thermodynamic and a kinetic one.
Fundamental features in the thermodynamic methods are the use of a suitable protease for catalyzing the establishment of the thermodynamic equilibrium between the reactants, and a removal of the reaction product from the reaction mixture. Thus, Isowa et al. (ref. 7-9) and U.S. Pat. No. 4,119,493 and British Pat. Nos. 1,523,546 and 1,533,129, Luisi et al. (ref. 12, 18) and Morihara (ref. 14) found that several serine, thiol and metalloendoproteases catalyze the synthesis of peptides from protected, but very easily soluble di-, tri- and tetrapeptides, provided the final products precipitate from the reaction mixture on account of their solubility being lower than the equilibrium concentration. Examples of such syntheses are illustrated in the following reaction scheme ##STR1##
However, this reaction principle can only be used as general method of synthesis if, like in the conventional chemical coupling procedure, all the amino side chains with potentially ionizable groups are blocked before the reaction and deblocked after the reaction. The method is also deficient in that the high specificity of the various enzymes calls for the use of various enzymes, depending upon the type of the peptide bond to be synthetized, or rather of the amino acids forming the peptide bond. Even though these complications might be overcome, the method involves several other problems because it requires a very high concentration of enzyme (100 mg/mmole peptide), long reaction periods (1 to 3 days) and gives very varying yields (typically 20 to 80%, cf. the above mentioned U.S. and British patents).
Klibanov et al. (ref. 10) have proposed to work in a system consisting of water and an organic solvent immiscible with water. This procedure, too, requires a high concentration of enzyme and a long reaction period of up to several days.
The other type of enzymatic syntheses rely on the kinetic approach of the reaction. It has been shown for most serine and thiol proteases that an acyl enzyme intermediate is formed in one of the catalytic steps during the hydrolysis of peptides or peptide esters, which is then hydrolyzed by water in the subsequent step or steps. If other nucleophiles than water are present during the hydrolysis, they too will accept the acyl group from the acyl enzyme, resulting in the formation of an amide bond. This has been studied e.g. by Fastrez and Fersht (ref. 3) who report chymotrypsin-catalyzed hydrolysis of N-acetyl-L-phenylalanine ethyl ester (Ac-Phe-OEt) in the presence of various amino acid amides. The reaction is shown in scheme (4): ##STR2##
First an enzyme substrate complex is formed followed by the formation of the acyl enzyme intermediate (Ac-Phe-CT). This is hydrolyzed by water to Ac-Phe-OH, but if a nucleophile (R--NH.sub.2) is also present, the acyl enzyme intermediate will be subject to aminolysis in addition to hydrolysis. Assuming that k.sub.3 &gt;&gt;k.sub.-3 and k.sub.4 &gt;&gt;k.sub.-4, the ratio of aminolysis to hydrolysis clearly depends on k.sub.4 /k.sub.3 as well as on the concentration of the nucleophile which is in competition with 55 M water. Fastrez and Fersht found that e.g. 1 M alanine amide at pH 10 is a 44 times stronger nucleophile than 55 M water, which resulted in a predominant formation (larger than 95%) of the shown N-acyldipeptide-amide. Morihara and Oka (ref. 13-16) have further exploited this principle for enzymatic peptide synthesis. Using chymotrypsin and trypsin they synthetized a plurality of peptides from N-acylamino acid esters and amino acid derived nucleophiles and demonstrated that the reaction was purely kinetic since high yields could be obtained, independent of the solubility of the product.
The studies mentioned above seem to indicate that kinetic approaches possess several advantages over the thermodynamic methods:
1. Quicker reaction (in certain cases completed within few minutes).
2. Possibility of using low concentrations of enzyme.
3. Amino acid side chains must not necessarily be blocked.
4. Immobilized and insoluble enzymes may be used as the peptides are in solution, permitting automatization.
As, however, the reaction products may be soluble the synthesis must be quicker than the secondary hydrolysis of the product so that they can be separated in time.
Moreover, the known kinetic approaches are limited in their applicability because the specific properties of the enzymes examined call for the use of various enzymes for the synthesis of the various peptide bonds. Additionally, synthesis of large peptide molecules invariably causes the internal peptide bonds in the molecule to be hydrolyzed, independent of the esterolytic activity of the enzymes, due to the endopeptidase activity of the enzymes used till now.